Project Summary Rotaviruses (RVs) are a medically important human pathogen and the predominant cause of severe gastroenteritis, vomiting, and diarrhea in infants and young children worldwide. RVs are also a great model to interrogate the antiviral responses at the host mucosal surfaces. Our overall objectives are to better understand RV-host interactions and to use that information to develop improved RV vaccines and therapeutic interventions, thereby preventing and treating enteric virus infections. The host interferon (IFN) signaling underlies the basis of RV host range restriction and suppresses the replication of RVs not native to that species in vivo. However, the specific IFN-mediated antiviral effectors are not known and the associated molecular mechanisms remain unclear. To bridge this gap in knowledge, we sought to define the most highly induced IFN-stimulated genes (ISGs) in primary human intestinal epithelial cells (IECs). Using an IEC-specific ISG gain-of-function screening approach, we identified several novel host factors that restrict RV replication, including sterile alpha motif domain-containing 9 (encoded by SAMD9). Intracellular viral RNA levels and virus progeny production were significantly enhanced in SAMD9 CRISPR knockout cells. In parallel, we also made the exciting discovery that RV encodes non-structural protein 1 (NSP1) to target SAMD9 for proteasomal degradation. In this R01 application, using a set of novel, powerful, and tractable model systems, we will test the hypotheses that SAMD9 confines early RV replication in an epithelial cell-specific manner and that RV NSP1 functions to overcome SAMD9 restriction to promote viral replication and pathogenesis in vivo. In Aim 1, we will examine the mechanistic basis underlying SAMD9 inhibition of RV replication in vitro using several newly available fluorophore-labeled RVs and a recently developed RV reverse genetics system. We will test these findings in a physiologically relevant human small intestinal organoid culture derived from healthy individuals and SAMD9-mutation patients. In Aim 2, we will examine how RV NSP1 binds to SAMD9 via a novel recognition motif and induces its degradation. We will determine whether NSP1 degrades SAMD9 in IECs in vivo and if this process contributes to successful RV intestinal replication using a novel neonatal rat model. Collectively, we expect these studies on SAMD9-RV interactions to have a substantial impact on elucidating the basic biology of ISG mode of action, identifying new viral innate immune evasion mechanisms, and laying the scientific foundation for the rational design of new RV vaccine candidates based on targeted NSP1 attenuation.